Folding Features and Dynamics of 3D Genome Architecture in Plant Fungal Pathogens

ABSTRACT The folding and dynamics of three-dimensional (3D) genome organization are fundamental for eukaryotes executing genome functions but have been largely unexplored in nonmodel fungi. Using high-throughput sequencing coupled with chromosome conformation capture (Hi-C) data, we generated two chromosome-level assemblies for Puccinia striiformis f. sp. tritici, a fungus causing stripe rust disease on wheat, for studying 3D genome architectures of plant pathogenic fungi. The chromatin organization of the fungus followed a combination of the fractal globule model and the equilibrium globule model. Surprisingly, chromosome compartmentalization was not detected. Dynamics of 3D genome organization during two developmental stages of P. striiformis f. sp. tritici indicated that regulation of gene activities might be independent of the changes of genome organization. In addition, chromatin conformation conservation was found to be independent of genome sequence synteny conservation among different fungi. These results highlighted the distinct folding principles of fungal 3D genomes. Our findings should be an important step toward a holistic understanding of the principles and functions of genome architecture across different eukaryotic kingdoms. IMPORTANCE Previously, our understanding of 3D genome architecture has mainly come from model mammals, insects, and plants. However, the organization and regulatory functions of 3D genomes in fungi are largely unknown. In this study, we comprehensively investigated P. striiformis f. sp. tritici, a plant fungal pathogen, and revealed distinct features of the 3D genome, comparing it with the universal folding feature of 3D genomes in higher eukaryotic organisms. We further suggested that there might be distinct regulatory mechanisms of gene expression that are independent of chromatin organization changes during the developmental stages of this rust fungus. Moreover, we showed that the evolutionary pattern of 3D genomes in this fungus is also different from the cases in mammalian genomes. In addition, the genome assembly pipeline and the generated two chromosome-level genomes will be valuable resources. These results highlighted the unexplored distinct features of 3D genome organization in fungi. Therefore, our study provided complementary knowledge to holistically understand the organization and functions of 3D genomes across different eukaryotes.

In this study, Xia et al. employed Hi-C to reconstruct the chromatin interaction maps in Puccinia striiformis f. sp. Tritici (Pst), a plant pathogenic fungus that causes wheat rust disease. With these data, the authors discovered that wheat rust fungi adopt Rabl configuration and their genomes are organized into distinct globule models (fractal vs equilibrium) on different scales. By comparing the genome organization and gene expression between different growth stages, they found gene regulation is independent of 3D structure of the genome. Then the authors investigated if chromosome conformation at syntenic regions is conversed between species. Unexpectedly, distinct from mammalian genomes, 3D chromatin conformation is not conserved between filamentous fungi even at syntenic regions. The authors also discovered that A/B compartments seem not exist in wheat rust fungi, suggesting a very different genome organization model in these species. I found this manuscript is well organized and provides useful resources as well as solid conclusions. Below are some comments that in my opinion, may help improve this paper.
1. First paragraph in RESULTS. As a non-expert in wheat rust fungi, I had no idea what CYR34 or 93-210 was until I found some introduction to them in MATERIALS AND METHODS. It's better to move these words into the main text before presenting the data.
2. Fig 1. I understand no Pst genome had been successfully assembled before this study, so it's fundamental to describe the assembly of the 2 Pst genomes (CYR34 and 93-210) before presenting the Hi-C data. However, I don't think it's necessary to show Fig 1 in the main text. It's not related to the main theme of this study ---genome organization. Besides, panel A is difficult to read. It may be a good idea to move it into supplemental materials.
3. Paragraph starting at line 154. The 2009 Science paper by Liberman-Aiden (the first Hi-C paper in which globule models were explained) should be cited. This analysis is inappropriate and does not support the conclusion that "fold changes of gene expression in these two regions had no significant difference". The authors cherry-picked only the affected genes. It's very likely that there will be no significant difference if up-regulated genes in dis-similar regions are compared to up-regulated genes in conservation regions. I think the right way to do this is to 1) analyze all the genes in the dis-similar regions and see how their expression level changes between different growth stages (i.e. whether or not these genes tend to be up-or down-regulated); 2) do the same to all the genes in the conservation regions and see if they display the same tendency as in the dis-similar regions.
6. The "Avirulence gene cluster" paragraph. The description is too general. Fig 6B and E, please use arrows to indicate the centromere regions.
7. There are a bunch of grammar mistakes in the manuscript. It's helpful to turn on the grammar check function in Microsoft Word.
Reviewer #3 (Public repository details (Required)): All sequencing results are available via SRA. However I couldn't find the final genome assemblies. I think they are typically submitted to genbank? Or did I miss them somewhere?
Reviewer #3 (Comments for the Author): In this paper the authors assembled genomes of two wheat rust fungi, using short and long read seaquencing, and also Hi-C data. Then they used this Hi-C data to analyse the 3D organization of chromatin in these fungi, and to compare it to gene expression and synteny.
Overall this is an interesting study shedding light on some unique features of 3D organization in fungi. However I have a few important comments for the authors to consider regarding analysis and interpretation of Hi-C data.
1. First of all, when authors analyse the P(s) curves they only think in terms of fractal vs equilibrium globules, which is a highly simplified approach to chromatin structure. Do the authors think there is loop extrusion occurring in these species? From the flatter part in shorter distanced and a sharper drop later, I would expect so, this should be mentioned and discussed.
2. Why do inter-chromosomal contacts vary by distance? There are no distances, since regions are on different chromosomes there, so it is typically depicted as a single value.
3. Specifically the CYR34 germtube sample appears particularly interesting. To my knowledge, decay of contact frequency with the exponent of -2.7 has never been observed. This represents some highly unusual chromatin folding, unlikely to be any sort of globule even (certainly not equilibrium globule, as suggested by authors). Is there anything special going on with nuclear organization at this stage? Note that on the line 156 there is a typo: s^-3/2 corresponds to an equilibrium globule, not fractal globule.
4. Related to the same figure, the 3D modelling performed by the authors is incorrect. The tool is they used is generally designed for single-cell Hi-C, not bulk Hi-C like here. Interestingly, the tool does allow to process bulk data, but requires an argument -p that the authors did not use. In general, generating 3D structure from population/bulk Hi-C doesn't make much sense in my opinion (since it's averaged from millions of different conformations), and I would simply remove this part. 5. Regarding compartment analysis, I see some some misconception there. First of all, the sign of the eigenvector on its own is meaningless. Typically, the compartment signal is obtained by ensuring positive correlation of the first (or another top) eigenvector with something that would correlate with "active" regions. For example, in mammals GC content works very well, since genes have higher GC content than genomic average. Gene density of RNA-seq signal should work well in any organism with compartments. Then the eigenvector that has the highest absolute correlation with that track is chosen as the one that most likely corresponds to compartments, and in case the sign of the correlation coefficient is negative, the values of the eigenvector are flipped, so this way the regions with positive signs correspond to active regions.
6. In case the compartments are very weak and absent, the eigenvectors don't pick up eu-vs heterochromatin. They would pick up some other major features of the contact map, very often the centromeres vs chromosome arms (as in this case), or sometimes perhaps left arm vs right arm... So I wouldn't draw any conclusions about the chromatin state based on the eigenvectors in case with no visible compartments (of if the eigenvector for some reason didn't pick out the compartment structure).
7. Would be interesting to see some speculations about why only one of the fungi analysed here displays clear compartments, but it's understandable if authors think there is not enough known to even think about it here.
Finally, the writing is completely understandable, but would benefit from proofreading to remove occasional typos or grammatical errors.

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In this manuscript, Xia et al generate two chromosome-level genome assemblies for a plant fungi, pst, using Hi-C assisted scaffolding pippeline and investigated the chromosome conformations of this organism and a few other fungi. In general, the manuscript is clearly written and I have only a few minor points for the author to consider.
Line 41 & 42, it was suggested that the regulation of gene activities might be independent of the changes of genome organization. However, in line 52 & 53, the author suggest that 3D genome features might contribute to the regulatory mechanism of gene expression. Please clarify these conclusions.
Could the authors discuss possible reasons/factors that contribute to the formation of compartments in certain fungi, i.e. Epichloe festucae? Does this organism encode extra SMC family proteins compared to other fungi?
Minor comments.
Line 38, it should be "the fungi". 'the' is omitted in many sentences throughout the manuscript. Line 55, different from? Line 82, 'beside these model fungi', please check the use of "beside". Line 91, gene express Line 115, a brief introduction/description about the experiment setup is recommended, albeit it has been mentioned in the method part. Line 126, please specify the "LTR elements and DNA elements" Line 154, 'examined and plotted' should be changed to 'examine and plot' Line 186, genomic locations Line 194, Does this conclusion in the title apply to all studied fungal? Line 249, in my understanding, the last paragraph of the Results section seems belonging to another story. Line 261, together with data of other wheat rust fugal Line 302, compartmentalization was not observed in all species. Line 316, The low expression in peri-centromeric heterochromatin regions could also be a result, but not a driver for EHE formation. Line 358 & Line 359, 'Shacking' should be changed to 'shaking' Line 422, 'A genome' Line 452, do you mean 'active or inactive'?

Reviewer comments:
We thank all reviewers for their constructive comments and suggestions to improve the quality and clarity of the manuscript. Bellow are our responses to each point of the comments or suggestions.

Reviewer #1 (Public repository details (Required)):
the NGS data should be deposited in a public repository. Response: Thanks. To make the data repository clear, we have added accession numbers in the "Data availability" section.

Reviewer #1 (Comments for the Author):
In general, the manuscript is clearly written and I have only a few minor points for the author to consider. Response: Thanks for the positive feedback. Every suggestion is highly appreciated and considered in our revision.

Line 41 & 42, it was suggested that the regulation of gene activities might be independent of the changes of genome organization. However, in line 52 & 53, the author suggest that 3D genome features might contribute to the regulatory mechanism of gene expression. Please clarify these conclusions.
Response: Thanks for pointing out this misleading. To clarify this, we changed the statement as "There might be distinct regulatory mechanisms of gene expression that are independent of changes of chromatin organization during developmental stages of rust fungi." (Lines 57-58)

Could the authors discuss possible reasons/factors that contribute to the formation of compartments in certain fungi, i.e. Epichloe festucae? Does this organism encode extra SMC family proteins compared to other fungi?
Response: We obtained the annotated proteomes of 8 fungi, and searched for the presence of PF02463, the domain at the N terminus of SMC proteins. Unfortunately, it seems the numbers of SMC proteins do not have much difference in the examined fungi.
In fact, the mechanism underlying the formation of compartments is still unclear. The most updated idea is that formation of compartment is mediated by homotypic interactions among genome regions with the same transcriptional state, and then generated phase-separated structures. Based on this, we could only provide some thinking on possible reasons why compartment is present in certain fungi but not others. We have added sentences to discuss the point (Lines 348-360).